Search PubMed⌕ Search

Biomedical subjects

B Moss

Publications and source records attributed to B Moss.

At least 217 records · Page 12Linked to original sources
PubMed1991

Identification, sequence, and expression of the gene encoding a Mr 35,000 subunit of the vaccinia virus DNA-dependent RNA polymerase.

The gene rpo35, encoding a subunit of the vaccinia virus DNA-dependent RNA polymerase, was identified, and its RNA and protein products were characterized. An Mr 35,000 polypeptide, which bound antibody to the purified RNA polymerase, was synthesized in reticulocyte lysates programmed with viral mRNA that hybridized to a 2,300-base pair segment of the viral genome. Determination of the sequence of the DNA segment revealed four potential protein coding regions, none of which had evident similarity to any described RNA polymerase subunit of prokaryotes or eukaryotes. One open reading frame that could encode a 35,400-Da protein was identified as rpo35 on the basis of mRNA hybridization, cell-free translation, and immunoprecipitation. The identification was confirmed by sequencing tryptic peptides of the authentic Mr 35,000 RNA polymerase subunit. Antiserum to the purified recombinant protein, expressed in bacteria, reacted specifically with a Mr 35,000 polypeptide that was detected starting 2 h after virus infection and that co-sedimented with RNA polymerase purified from virions. RNA analyses indicated that the 5'-end of an early transcript started 25 nucleotides upstream of rpo35, which is consistent with the location of an early promoter consensus sequence.

Amino Acid Sequence↗
PubMed1991

Heterologously expressed serotonin 1A receptors couple to muscarinic K+ channels in heart.

In cardiac atrial cells, muscarinic acetylcholine receptors activate a K+ current directly via a guanine nucleotide-binding protein (G protein). Serotonin type 1A receptors may activate a similar pathway in hippocampal neurons. To develop a system in which receptor/G protein/K+ channel coupling can be experimentally manipulated, we have used a highly efficient recombinant vaccinia virus vector system to express human serotonin 1A receptors in primary cultures of rat atrial myocytes. The expressed 1A receptors activated the inwardly rectifying K+ conductance that is normally activated by the endogenous muscarinic acetylcholine receptors. Maximal responses to either agonist occluded further activation by the other agonist. The average activation time constants for serotonin were about 5 times slower than for acetylcholine. The data support suggestions that the intracellular signaling pathway from seven-helix receptors to G proteins and directly to ion channels is widespread in excitable cells. After a fraction of the G proteins are activated irreversibly by guanosine 5'-[gamma-thio]triphosphate, subsequent transduction proceeds more efficiently. One possible interpretation is that multiple G-protein molecules are required to activate each channel. Vaccinia virus expression vectors are thus useful for expressing seven-helix receptors in primary cultures of postmitotic cells and have provided a heterologous expression system for the signaling pathway from seven-helix receptors to G proteins and directly to ion channels.

Acetylcholine↗
PubMed1991

Vaccinia virus: a tool for research and vaccine development.

Vaccinia virus is no longer needed for smallpox immunization, but now serves as a useful vector for expressing genes within the cytoplasm of eukaryotic cells. As a research tool, recombinant vaccinia viruses are used to synthesize biologically active proteins and analyze structure-function relations, determine the targets of humoral- and cell-mediated immunity, and investigate the immune responses needed for protection against specific infectious diseases. When more data on safety and efficacy are available, recombinant vaccinia and related poxviruses may be candidates for live vaccines and for cancer immunotherapy.

Animals↗
PubMed1991

Induction of Plasmodium falciparum transmission-blocking antibodies by recombinant vaccinia virus.

Many candidate antigens of malaria vaccines have limited immunological recognition. One exception is Pfs25, a cysteine-rich, 25-kilodalton sexual stage surface protein of Plasmodium falciparum. Pfs25 is a target of monoclonal antibodies that block transmission of malaria from vertebrate host to mosquito vector. The surface of mammalian cells infected with a recombinant vaccinia virus that expressed Pfs25 specifically bound transmission-blocking monoclonal antibodies. Furthermore, major histocompatibility complex-disparate congenic mouse strains immunized with recombinant Pfs25 elicited transmission-blocking antibodies, demonstrating that the capacity to develop transmission-blocking antibodies is not genetically restricted in mice. Live recombinant viruses may provide an inexpensive, easily administered alternative to subunit vaccines prepared from purified recombinant proteins to block transmission of malaria in developing countries.

Animals↗
PubMed1991

Inducer-dependent conditional-lethal mutant animal viruses.

Regulatory elements of the Escherichia coli lac operon were used to construct an inducer-dependent conditional-lethal mutant animal virus. The gene encoding the repressor protein of the lac operon was integrated into the vaccinia virus genome so that it was expressed constitutively, and the lac operator was inserted next to the promoter of a gene that encodes an 11-kDa virion-associated protein of unknown function. The addition of inducer to the cell culture medium provided permissive conditions for isolation of a conditional-lethal mutant virus. Under nonpermissive conditions, the isolated virus did not form plaques, and the yield was decreased by at least 1000-fold under one-step growth conditions. Transcription of the operator-controlled gene was inducer-dependent and necessary for synthesis of the 11-kDa protein. Application of this mutagenesis strategy to other viruses is discussed.

Animals↗
PubMed1991

The assembly of the HIV-1 env glycoprotein into dimers and tetramers.

The envelope (env) glycoprotein of human immunodeficiency virus 1 (HIV-1), initially synthesized as a precursor molecule termed gp160, is cleaved into two noncovalently associated subunits prior to delivery to the plasma membrane. We have studied the oligomeric structure of this protein using chemical cross-linking, velocity gradient sedimentation, and SDS-resistance. We find that gp160 forms stable homodimers after synthesis. After cleavage to gp120/gp41 the molecule becomes less stable to detergent solubilization and centrifugation but remains dimeric. Interactions between the 129 amino terminal residues in the ectodomains of adjoining gp41 subunits are both sufficient and necessary for assembly. In addition, tetramers composed of two dimers were also formed. Larger structures were not observed. The tetrameric paramyxovirus F protein, which has structural and functional similarities to the HIV-1 env protein, also forms a dimer of dimers.

Gene Products, env↗
PubMed1991

Identification, sequence, and expression of the gene encoding the second-largest subunit of the vaccinia virus DNA-dependent RNA polymerase.

The gene, rpo 132, encoding the second-largest subunit of the vaccinia virus DNA-dependent RNA polymerase was identified and sequenced. Two complementary approaches, involving antiserum to purified vaccinia virus RNA polymerase, were used to locate the rpo 132 gene. One method involved the screening of a lambda gt11 library of vaccinia virus genome fragments and the other was based on the immunoprecipitation and polyacrylamide gel electrophoresis of the in vitro translation products of mRNA that hybridized to immobilized vaccinia virus DNA. The deduced open reading frame of the rpo 132 gene predicted a polypeptide of 1164 amino acid residues with sequence similarities to the second-largest RNA polymerase subunits of eubacteria, archaebacteria, and eukaryotes as well as to other poxviruses. Transcriptional analyses indicated that rpo 132 has both early and late RNA start sites and is expressed throughout infection.

Amino Acid Sequence↗
PubMed1991

Vaccinia virus-mediated expression and identification of the human poliovirus receptor.

We have used cDNA encoding the cellular receptor for poliovirus (PVR) to prepare polyclonal antisera against beta-galactosidase PVR fusion proteins. One of these antisera allowed identification of a glycoprotein doublet band of about 67 kDa in membrane preparations of HeLa cells and in a PVR cosmid-bearing mouse cell line. In vitro translation of PVR-specific transcripts gave rise to a protein of 46 kDa; the product had a molecular weight of 67 kDa when microsomal membranes were added to the cell-free extract. Overexpression of PVR cDNA in mouse L-cells by means of a recombinant vaccinia virus led to the synthesis of a glycoprotein having a molecular weight identical to that of the glycosylated in vitro product. The vaccinia virus-mediated protein was also recognized by a monoclonal antibody that blocks poliovirus infection. Its biological activity was demonstrated by poliovirus binding and infectivity assays. The data show that PVR is a glycoprotein of 67 kDa and that this protein is sufficient to confer poliovirus susceptibility to mouse cells.

Animals↗
PubMed1991

Studies using a recombinant vaccinia virus expressing the circumsporozoite protein of Plasmodium berghei.

A recombinant vaccinia virus was constructed which expressed the circumsporozoite protein of Plasmodium berghei. Four different strains of mice belonging to different haplotypes were immunized with the recombinant virus. The antibody response to the circumsporozoite protein as well as to vaccinia virus varied among the strains, independently of each other. The anti-circumsporozoite protein titers were comparable to that obtained on immunization with irradiated sporozoites. Spleen cells from H2d mice immunized with P. berghei sporozoites showed a significant proliferative response when cultured in vitro with a low multiplicity of the recombinant vaccinia virus. A weak cytotoxic T lymphocyte response specifically targeting the circumsporozoite protein could be identified in spleens of BALB/c (H2d) mice immunized with vaccinia virus when BALB 3T3 cells transformed with a plasmid expressing the circumsporozoite protein under control of the simian virus 40 promoter were used as target cells in the cytotoxic T lymphocyte assay. However, none of the recombinant virus-immunized animals could be protected from a challenge of sporozoites even at the lowest dose of parasite used.

Animals↗
PubMed1991

Anti-HIV activity of CD4-Pseudomonas exotoxin on infected primary human lymphocytes and monocyte/macrophages.

CD4(178)-PE40 is a recombinant protein consisting of a portion of human CD4 linked to active domains of Pseudomonas aeruginosa exotoxin A. In previous experiments with human T cell lines, the hybrid toxin was found to selectively kill cells infected with human immunodeficiency virus type 1 (HIV-1), and to inhibit HIV-1 spread in mixtures of infected and uninfected cells. CD4(178)-PE40 inhibits HIV-1 spread in cultured primary human lymphocytes. Moreover, the hybrid toxin selectively kills HIV-1 chronically infected monocyte/macrophage cell lines and inhibits HIV-1 spread in primary macrophage cultures. Control experiments indicate that the protective effects of CD4(178)-PE40 against HIV-1 spread are due to selective killing of the infected cells rather than simply to neutralization by the CD4 moiety. Thus, for the major cell types susceptible to HIV infection in vivo, surface envelope glycoprotein is expressed at sufficient levels to enable binding and internalization of CD4(178)-PE40 and consequent selective cell killing.

Bacterial Proteins↗
PubMed1991

Expression of ion channels and receptors in Xenopus oocytes using vaccinia virus.

The cytoplasmic injection of mRNA synthesized in vitro into Xenopus oocytes is widely used for heterologous expression of ion channels and neurotransmitter receptors. We report two new methods for expression of ion channels and receptors in oocytes using vaccinia virus (VV). 1) A recombinant VV carrying the Shaker H4 K+ channel cDNA driven by the VV P7.5 early promoter was injected into oocytes. 2) A recombinant VV containing the bacteriophage T7 RNA polymerase driven by the P7.5 promoter was coinjected along with plasmids containing a T7 promoter and cDNAs for channels and receptors. The functionally expressed proteins include a) voltage-gated ion channels: the Shaker H4 K+ channel and the rat brain IIA Na+ channel, b) a ligand-gated ion channel: the mouse muscle nicotinic acetylcholine receptor (AChR), and c) a G protein-coupled receptor: the rat brain 5HT1C receptor. After virus/cDNA injection into oocytes, these channels and receptors generally showed characteristics and expression levels similar to those observed in mRNA-injected oocytes. However, the AChR expressed at lower levels in virus/cDNA-injected oocytes than in mRNA-injected oocytes. Because our methods bypass mRNA synthesis, they are more rapid and convenient than the mRNA injection method. Potential applications to structure-function studies and expression cloning are discussed.

Animals↗
PubMed1991

Biological and immunological properties of human immunodeficiency virus type 1 envelope glycoprotein: analysis of proteins with truncations and deletions expressed by recombinant vaccinia viruses.

The effects of C-terminal and internal deletions on the synthesis, transport, biological properties, and antigenicity of the human immunodeficiency virus type 1 envelope protein were determined. A family of recombinant vaccinia viruses that express N-terminal overlapping env proteins of 204, 287, 393, 502 (full-length gp120), 635, 747, and 851 (full-length gp160) amino acids was constructed. All of the proteins were detected in intra- and extracellular forms which differed in the extent of glycosylation. The 747- and 851-amino-acid proteins were cleaved, were expressed on the surface of infected cells, and bound CD4. The 635-amino-acid env protein was cleaved inefficiently, and both the precursor and product were secreted, indicating absence of the transmembrane sequence. The 635- as well as the 502-amino-acid protein, which was also largely secreted, could still bind CD4. Unexpectedly, the 393-amino-acid protein was anchored in the plasma membrane, but neither it nor smaller proteins bound to soluble CD4. When amino acids at the gp120-gp41 junction were deleted, proteolytic cleavage of gp160 did not occur. Nevertheless, gp160 was inserted into the plasma membrane and bound soluble CD4. The predominant conserved B-cell epitopes were mapped to gp41 and the C terminus of gp120, whereas cytotoxic T-cell epitopes were distributed throughout the length of the glycoproteins.

Animals↗
PubMed1991

Extracellular vaccinia virus formation and cell-to-cell virus transmission are prevented by deletion of the gene encoding the 37,000-Dalton outer envelope protein.

There are two types of infectious vaccinia virus particles: intracellular naked virions and extracellular enveloped virions (EEV). To determine the biological role of the enveloped form of vaccinia virus, we produced and characterized a mutant that is defective in EEV formation. The strategy involved replacement by homologous recombination of the gene F13L, encoding a 37,000-Da protein (VP37) that is specific for the outer envelope of EEV, with a selectable antibiotic resistance marker, the Escherichia coli gpt gene. Initial experiments, however, suggested that such a mutation was lethal or prevented plaque formation. By employing a protocol consisting of high-multiplicity passages of intracellular virus from the transfected cells and then limiting dilution cloning, we succeeded in isolating the desired mutant, which was defective in production of plaques and extracellular virus but made normal amounts of intracellular naked virions. Electron microscopic examination indicated that the mutant virus particles, unlike wild type, were neither wrapped with Golgi-derived membranes nor associated with the cell surface. The absence of VP37 did not prevent the transport of the viral hemagglutinin to the plasma membrane but nevertheless abrogated both low-pH- and antibody-mediated cell fusion. These results indicate that VP37 is required for EEV formation and also plays a critical role in the local cell-to-cell transmission of vaccinia virus, perhaps via enveloped virions attached to or released from the cell membrane. By contrast, a mutated virus with a deletion of the K4L open reading frame, which is a homolog of the VP37 gene, was not defective in formation of plaques or EEV.

Animals↗
PubMed1991

Vaccinia virus morphogenesis is interrupted when expression of the gene encoding an 11-kilodalton phosphorylated protein is prevented by the Escherichia coli lac repressor.

A conditional lethal vaccinia virus mutant, which constitutively expresses the Escherichia coli lac repressor and has the lac operator controlling the F18R gene (the 18th open reading frame of the HindIII F fragment of the vaccinia virus strain WR genome) encoding an 11-kDa protein, was previously shown to be dependent on the inducer isopropyl-beta-D-thiogalactoside (IPTG) for replication (Y. Zhang and B. Moss, Proc. Natl. Acad. Sci. USA 88:1511-1515, 1991). Further studies indicated that the yield of infectious virus could be regulated by titration with IPTG and that virus production was arrested by IPTG removal at appropriate times. Under nonpermissive conditions, an 11-kDa protein reactive with antiserum raised to a previously described DNA-binding phosphoprotein (S. Y. Kao and W. R. Bauer, Virology 159:399-407, 1987) was not synthesized, indicating that the latter is the product of the F18R gene. In the absence of IPTG, replication of viral DNA and the subsequent resolution of concatemeric DNA molecules appeared normal. Omission of IPTG did not alter the kinetics of early and late viral protein synthesis, although the absence of the 11-kDa polypeptide was noted by labeling infected cells with [35S]methionine or [32P]phosphate. Pulse-chase experiments revealed that proteolytic processing of the major viral structural proteins, P4a and P4b, was inhibited under nonpermissive conditions, suggesting a block in virus maturation. Without addition of IPTG, the failure of virus particle formation was indicated by sucrose gradient centrifugation of infected cell lysates and by the absence of vaccinia virus-mediated pH-dependent cell fusion. Electron microscopic examination of infected cells revealed that immature virus particles, with aberrant internal structures, accumulated when synthesis of the 11-kDa DNA-binding protein was prevented.

Animals↗
PubMed1991

Folding, interaction with GRP78-BiP, assembly, and transport of the human immunodeficiency virus type 1 envelope protein.

A detailed kinetic and quantitative analysis of the early and late biosynthetic events undergone by the human immunodeficiency virus type 1 envelope protein expressed by a recombinant vaccinia virus was performed. Early folding events that occurred in the endoplasmic reticulum included disulfide bond formation (t1/2 approximately 10 min), folding of envelope protein into a form competent to bind CD4 (t1/2 approximately 15 min), and specific and transient association and dissociation with GRP78-BiP (t1/2 approximately 25 min). After initial folding, envelope protein monomers formed noncovalently associated dimers with high efficiency (t1/2 approximately 30 min). Studies with brefeldin A, a compound that inhibits endoplasmic reticulum-to-Golgi transport, suggested that assembly occurred in the endoplasmic reticulum while cleavage of gp160 into gp120/gp41 subunits occurred in a post-endoplasmic reticulum compartment. Transport to the Golgi was monitored by modification of N-linked sugars to forms partially resistant to endoglycosidase H. The kinetics of endoglycosidase H resistance were nearly identical to the kinetics of gp160 cleavage (t1/2 approximately 80 min). Cleavage efficiency was strongly cell type dependent, ranging from 13 to 70%. By contrast, approximately 50% of the gp120 generated by the cleavage event was shed (t1/2 approximately 120 min) regardless of the cell type used. The results are discussed in terms of the overall biosynthetic pathway of the envelope protein and provide a framework with which to assess the effects of mutations on structure and function.

Acetylglucosaminidase↗
PubMed1991

A transcription factor for expression of vaccinia virus late genes is encoded by an intermediate gene.

A factor, designated VLTF-1, that is required in vitro for specific transcription of vaccinia virus late genes was previously isolated from vaccinia virus-infected cells. Subsequent genetic experiments identified three vaccinia virus genes, encoding proteins of 17, 26, and 30 kDa, that together trans activate late gene expression in vivo. The purpose of this study was to determine whether VLTF-1 corresponded to one of the three trans activators. Toward this end, VLTF-1 was further purified, the trans-activator genes were expressed in Escherichia coli, and antisera were made to the native and recombinant proteins. Antibody to the 30-kDa recombinant protein reacted on Western immunoblots with a protein of approximately Mr 30,000 that cochromatographed and cosedimented with VLTF-1 activity from virus-infected cells. Conversely, antibody to purified VLTF-1 bound to products produced by in vitro transcription and translation of the open reading frame encoding the 30-kDa trans-activator protein. Both antisera depleted VLTF-1 activity and blocked late gene transcription by partially purified extracts of vaccinia virus-infected cells. Taken together, these data demonstrate that the 30-kDa trans activator comprises part, if not all, of VLTF-1 activity.

Blotting, Western↗
PubMed1991

An HLA-C-restricted CD8+ cytotoxic T-lymphocyte clone recognizes a highly conserved epitope on human immunodeficiency virus type 1 gag.

A unique epitope on the gag protein of human immunodeficiency virus type 1 (HIV-1), located at amino acid 145 to 150, has been mapped by using a CD8+ cytotoxic T-lymphocyte (CTL) clone. This epitope is highly conserved among 18 HIV-1 strains. The HIV-1 gag-specific human leukocyte antigen (HLA) class I-restricted CD8+ CTL clone was generated from fresh peripheral blood mononuclear cells of an HIV-seropositive donor by stimulation with gamma-irradiated allogeneic peripheral blood mononuclear cells in the presence of an anti-CD3 monoclonal antibody and recombinant interleukin-2. This gag-specific CTL clone killed autologous target cells infected with a recombinant vaccinia virus containing the gag gene of HIV-1 and target cells pulsed with an authentic p24gag construct expressed in Escherichia coli. Fine specificity was determined by using a panel of overlapping 30-amino-acid-long synthetic peptides and subsequently using smaller peptides to precisely map the CTL domain on p24. The epitope is on a highly conserved region, and it overlaps with a major B-cell epitope of gag. This CD8+ T-cell epitope is restricted by HLA-Cw3, which has not been previously identified as a restricting element for human CTL responses.

Amino Acid Sequence↗